Generally, optical spectroscopy allows to investigate the structure of matter on an atomic level. While linear spectroscopy mainly allows to investigate stationary states, non-linear, time-resolved methods allow to investigate dynamical behaviour as well. However, the well established time-resolved techniques such as transient absorption spectroscopy and transient diffraction spectroscopy are still limited to the measurement of quantum mechanical populations, while the underlying reason for any dynamical behaviour of a quantum mechanical system, namely the couplings between quantum mechanical states are not accessible by these spectroscopic methods.
In order to investigate the couplings between quantum mechanical states, the so-called coherent two-dimensional (2D) spectroscopy may be employed, to which the present invention relates. By systematically varying time delays between three exciting laser pulses and carrying out a complete measurement of the non-linear optical response one may directly obtain information about the couplings between quantum states, which then allows to study energy transfer processes in complex systems at a spatial resolution on a nanometer scale and with a time resolution on the order of femtoseconds.
The results of such measurements are usually presented in intuitively understandable 2D-spectra, in which the emission frequency of the system is plotted versus the excitation frequency. Intensities of this 2D-spectrum lying off the diagonal axis, i.e. intensities for which the excitation and emission frequency are different from each other are indicative of couplings and thus transfer between individual energy levels. These intensities are also termed “cross peaks” for obvious reasons. Using 2D-spectroscopy one obtains the complete spectroscopic information up to third order, as all conventional spectroscopic techniques such as transient absorption etc. are implicitly included.
While 2D-spectroscopy is by far the most common type of multi-dimensional spectroscopy, it is nevertheless possible to use a larger number of excitation pulses and to consequently generate higher dimensional spectra. For example, five excitation pulses have been used in order to generate a fifth order optical signal. In the following description, for simplicity, reference is made to 2D-spectroscopy only. However, it is to be understood that the principles introduced herein may also be employed for higher dimensional spectroscopy.
An illustrative example for 2D-spectra is shown in FIG. 1, which has been taken from T Brixner, J. Stenger, H M Vaswani, M Cho, R. E. Blankenship, and G. R. Fleming, “Two-dimensional spectroscopy of electronic couplings in photosynthesis”, Nature 434, 625 (2005). Diagrams a, b and c of FIG. 1 show the 2D-spectra of the Fenna-Mathews-Olson (FMO) bacterium chlorophyll a (BChl) protein of green suphur bacteria, which serves both as an antenna for collecting light energy and as a mediator for directing light excitations from the chlorosome atenna to the reaction center. In diagrams a to c of FIG. 1 the horizontal axis corresponds to the absorption frequency ωr and the vertical axis corresponds to the emission frequency ωt. The 2D-spectra of diagrams a to c correspond to population times T of 0 fs, 200 fs and 1,000 fs, respectively, where the “population time” refers to the time between the second and third excitation light pulses.
Diagram d shows the experimental linear absorption spectrum (solid black) and its theoretically modelled counter part (dashed line), where individual excitation contributions are also shown (dashed-dotted line). In diagrams e and f, simulations of 2D-spectra are shown for T32 200 fs (diagram e) and T=1,000 fs (diagram f). Off-diagonal features such as those labelled A and B are indicators of electronic coupling and energy transport.
The reason for the occurrence of the non-diagonal intensity peaks A and B (“cross peaks”) in the spectra is that the structure of components of the macro molecules to which peaks A and B correspond are “aware” of each other. More precisely, this means that the structure components are so close to each other that a mutual quantum mechanical coupling exists, and the pulse sequence measures transitions between the states. The intensity in the 2D-spectrum represents the probability that a photon having the frequency ωr is absorbed and is emitted after a population time T at a frequency ωt. Note that such information cannot be discerned from one-dimensional spectra, and the example may illustrate the usefulness of and unique information contained in 2D-spectra.
Optical 2D-spectroscopy is in a sense analogous to 2D-NMR, which is nowadays a standard method for structure analysis of molecules and which is used in practically any chemical laboratory using commercially available apparatuses. 2D-NMR is based on the coupling between nuclear spins of individual atoms and therefore reflects the molecular structure. As is understood from the above description of FIG. 1, however, optical 2D-spectroscopy is sensitive to couplings between full chromophores, i.e. couplings between large molecular units or functional groups. As such, 2D-spectroscopy may reflect supramolecular structures which are of paramount interest and importance for many technical and scientific applications, for example in the field of organic photovoltaics, natural and artificial photosynthesis complexes, quantum dot systems etc. In addition, using 2D-spectroscopy dynamical processes on an ultrafast time scale in the femtosecond regime are accessible, while NMR-spectroscopy is limited to a time resolution on the order of milliseconds.
For a more complete description of the principles of coherent 2D optical spectroscopy, reference is made to the review article “Two-dimensional femtosecond spectroscopy” of David M. Jonas, Annual Ref. Phys. Chem. 2003, Vol. 54, 425-463, and to the review article “Coherent two-dimensional optical spectroscopy” of Minhaeng Cho, Chem. Rev. 2008, 108: 1331-1418 and the references cited therein.
Ever since the primary works by Hamm et al. (S. Woutersen and P. Hamm. “Structure determination of trialanine in water using polarization sensitive two-dimensional vibrational spectroscopy”, Journal of Physical Chemistry B 104, 11316 (2000)), Hochstrasser et al. (P. Hamm, M H Lim, and R. M Hochstrasser, “Structure of the amide i band of peptides measured by femtosecond nonlinear-infrared spectroscopy”, Journal of Physical Chemistry B 102, 6123 (1998)), and Tokmakoff et al. (M. Khalil, N. Demirdoven, and A. Tokmakoff, “Coherent 2D IR spectroscopy: molecular structure and dynamics in solution”, Journal of Physical Chemistry A 107, 5258 (2003)), coherent optical 2D-spectroscopy in the IR regime can be regarded as a well established method. 2D-spectroscopy for electronic transitions in the near infrared at 800 nm has been developed by Jonas et al. (J. D. Hybl, A. W. Albrecht, S. M. G. Faeder, and D. M. Jonas, “Two-dimensional electronic spectroscopy”, Chemical Physics Letter).
In the implementation of the two-dimensional spectroscopy, generally a non-collinear so-called “box”-geometry is chosen. Herein, the non-linear third order signal, i.e. the non-linear signal generated in response to an interaction of the sample with three ultrashort laser pulses is completely characterized by interference with a fourth and known laser pulse, which is referred to as the local oscillator, LO. A prerequisite for this so-called heterodyne detection, however, is that the optical phases of the individual pulses are constant with respect to each other. In other words, the setup must ensure that no unintentional variations in the arrival times of the pulses at the sample location may occur.
Clearly, phase stability is the more difficult to achieve, the shorter the wavelength of the light signal is. For example, since the typical IR wavelength is about a factor of 10 longer than a wavelength in the visible spectrum, this means that in the IR regime, for the same phase stability 10 times larger variations in the optical paths can be tolerated, which for example could be introduced by vibrations of mirrors or other optical components. This is why in IR 2D-spectroscopy, generally no special means for phase stabilization are necessary.
In 2004, the 2D-spectroscopy method has been applied for shorter wavelengths in the visible spectral region by Brixner et al. (T. Brixner, I. V. Stiopkin, and G. R. Fleming, “Tunable two-dimensional femtosecond spectroscopy”, Optics Letters 29, 884 (2004); T. Brixner, T Man{hacek over (c)}al, I. V. Stiopkin, and G. R. Fleming, “Phase-stabilized two-dimensional electronic spectroscopy”, Journal of Chemical Physics 121, 4221 (2004)), and by Miller et al. (M. L. Cowan, J. P. Ogilvie, and R. J. D. Miller, “Two-dimensional spectroscopy using diffractive optics based phased-locked photon echoes”, Chemical Physics Letters 386, 184 (2004); V. I. Prokhorenko, A. Halpin, R. J. D. Miller, “Coherently-controlled two-dimensional photon echo electronic spectroscopy”, Optics Express 17, 9764 (2009)). The method of Miller et al. uses a diffractive optical element at which two beams are split into a total of four beams, where two of the beams are each stable in phase with respect to each other. The two initial beams are, however, generated in a conventional manner using transmissive beam splitters. Herein, time delays between the pulses are introduced using conventional delay lines and retroflectors or rotatible glass plates. The setup of Brixner et al. also uses a diffractive optical element, however, instead of conventional delay lines, pairs of slideable or movable glass wedges are used to precisely introduce the delays.
A further method for obtaining phase stability and for introducing precise delays have been suggested by Nelson et al. (T. Hornung, J. C. Vaughan, T. Feurer, and K. A. Nelson, “Degenerate four-wave mixing spectroscopy based on two-dimensional femtosecond pulse shaping”, Optics Letters 29, 2052 (2004)), and by Damrauer and Zanni (E. M Grumstrup, S.-H. Shim, M. A. Montgomery, N. H. Damrauer, and M. T. Zanni, “Facile collection of two-dimensional electronic spectra using femtosecond pulse-shaping technology”, Optics Express 15, 16681 (2007)). Herein, an active phase modulator is employed in a pulse shaping device.
FIG. 2 schematically shows a further prior art experimental setup proposed by the present inventors, where the figure has been cited from U Selig, F. Langhojer, F. Dimler, T. Löhrig, C. Schwarz, B. Giesking, T. Brixner, “Inherently phase-stable coherent two-dimensional spectroscopy using only conventional optics” Optics Letter 33, 2851 (2008). The same setup is also described in detail in German patent DE 10 2008 025 170. As can be seen in FIG. 2, the incoming beam is split by a first beam splitter (BS1) having a conventional metallic coating. The transmitted beam (dashed line) propagates in the drawing plane, and the reflected beam (dotted line) is redirected by mirror M1 into a parallel and vertically displaced plane. Both beams hit a second conventional metallic beam splitter BS2, now creating four beams, of which three are used as excitation pulses (pulses {circle around (1)}, {circle around (2)} and {circle around (3)}) and one is used as a local oscillator signal {circle around (4)} in a standard non-collinear box geometry after focussing with the spherical mirror via a folding mirror FM into the sample.
Time delays between the pulses are provided by two piezo stages, namely a stage DS1 for delaying pulse pair {circle around (3)}/{circle around (4)} versus pulse pair {circle around (1)}/{circle around (2)} and a stage DS2 for delaying pulse pair {circle around (1)}/{circle around (3)} versus pulse pair {circle around (2)}/{circle around (4)}. The heterodyne signal which corresponds to the interference signal of the third order optical signal due to interaction of pulses {circle around (1)}, {circle around (2)} and {circle around (3)} with a sample on the one hand and the fourth light pulse {circle around (4)} (i.e. the local oscillator) on the other hand passes through an aperture. It is collected with a microscope objective and then passed through a single-mode fibre to a CCD array spectrometer. Since only pulse pairs are delayed and no individual beam hits any individual mirror or beam splitter, an inherent phase stabilization is achieved.
The phase stabilization can be understood considering that the signal phase φs depends on the arrival times ti of the three excitation pulses as exp(iφS)˜exp(iω0(−t1+t2+t3)), and the phase of the oscillating part of the spectral interference fringe pattern is φSI=φLO−φS, where φLO is the phase of the fourth light pulse, i.e. the local oscillator signal. Any path length changes due to mirror vibrations etc. will result in variations Δti of the arrival times. In the setup of FIG. 2, the corresponding phase fluctuations ω0Δti are correlated in the sense that between beam splitters BS1 and BS2, one obviously has Δt1=Δt2 and Δt3=Δt4, because beams {circle around (1)} and {circle around (2)} as well as {circle around (3)} and {circle around (4)} have not been separated yet. The subsequent handling of beam pairs {circle around (1)}/{circle around (3)} and {circle around (2)}/{circle around (4)} leads to Δt1−Δt3 and Δt2=Δt4. Accordingly, the following equation holds:ω0(−Δt1+Δt2+Δt3−Δt4)=0,which means that the detected spectral interference fringes are stable. For a more detailed explanation and derivation of this phase stabilization, reference is made to section II of DE 10 2008 025 170, in particular to equations (1) through (8) and the corresponding explanation, which shall not be repeated here but is incorporated herein by reference.
In U. Selig, F. Langhojer, F. Dimler, T. Löhrig, C. Schwarz, B. Gieseking, T. Brixner, “Inherently phase-stable coherent two-dimensional spectroscopy using only conventional optics” Optics Letters 33, 2851 (2008), from which FIG. 2 is taken, it has been demonstrated that the setup provides indeed a remarkable phase stability and reproducibility. Remarkably, the inherent phase stability of the setup allows to completely dispense with diffractive optical elements, as the separation of the light beams is achieved solely by ordinary beam splitters BS1 and BS2. An advantage of avoiding a diffractive optical element is that a spatial chirp associated with the diffractive optical element, which becomes noticeable for larger band widths, can be avoided, as is explained in detail in DE 10 2008 025 170.
An article by Nemeth et. al. (Compact phase-stable design for single- and double-quantum two-dimensional electronic spectroscopy, Alexandra Nemeth, Jaroslaw Sperling, Jürgen Hauer, Harald F. Kauffmann and Franz Milota, to be published in Optics Letters) has been posted under http://www.opticsinfobase.org on Oct. 2, 2009. In this article, an optical assembly for 2D-spectroscopy is shown, in which a base light pulse is simultaneously split into four light pulses using a transmissive optical grating. The four light beams are parallelised using a combination of a flat mirror and a spherical mirror and are focused using a similar combination of a spherical mirror and a flat mirror. Three out of the four light pulses are individually delayed using pairs of transmissive glass wedges that are movable with respect to each other.
In spite of the enormous advantages of 2D-spectroscopy, from an experimental point of view, the technique is still rather difficult to implement. Until today no 2D-spectroscopy apparatuses are commercially available, instead, 2D-spectroscopy is still only practised by experts in the field of ultrafast spectroscopy. There is thus a need for an experimental setup that would be simple and stable enough to be used by a broader range of scientists with no particular expertise inultrafast optics.
While with the different advances in the field summarized above the transition from IR to visible optical spectra could be achieved, until now it has not been possible to carry out 2D-spectroscopy in the UV range, because the phase stability criterion becomes even more difficult to meet, as the wavelengths are again a factor of 2 or 2.5 smaller than in the visible range.
However, it would be most desirable to make 2D-spectroscopy also available in the UV regime, since this would allow to study quantum mechanical couplings in completely new classes of samples which in this regard have hardly been understood so far, such as proteins. Currently, electronical processes in proteins are usually investigated using fluorescence techniques and transient absorption. However, it has so far not been possible to investigate couplings in a time-resolved manner, while exactly this would be extremely interesting for many proteins. For example, ultrafast charge transfer processes in proteins are barely understood up to now, and neither are the ultrafast changes in the spatial conformation of highly delocalized excimeres in proteins. 2D-spectroscopy at wavelengths as low as 250 nm could be the key for understanding ultrafast protein dynamics of different types, such as charge transfer in cryptochromes, initial stimulation of bacteriorhodopsin and understanding the mechanisms responsible for the photo stability of DNA. Also, UV 2D-spectroscopy could allow to study aromatic amino acids, which are generally believed to be the key for understanding the photo chemistry of proteins. While in current 2D-spectroscopy in the visible range one is still dependent on the spectral position of suitable chromophores, 2D-spectroscopy in the ultraviolet regime would allow to study mostly any type of organic substances, their supra molecular configuration and their ultrafast dynamics.
A problem underlying the invention is to provide an optical assembly for 2D-sepctroscopy that is simple in construction and stable in operation. A further problem underlying the invention is to provide an optical assembly for a 2D-spectroscopy apparatus which is especially suitable for carrying out 2D-spectroscopy at wavelengths in the UV regime.